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Distinct roles of Central missing and Dispatched in sending the Hedgehog signal

Center for Developmental Biology and Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9133, USA

View larger version (23K): [in a new window]   Fig. 1. Tissue-specific mosaic screen for mutations in the hh pathway. (A) A two-step screen was designed to identify genes that positively regulate the hh pathway. In the primary screen, mosaic flies were generated using the eyFLP system and mutations that caused hh-like small-eye phenotypes were isolated in the F1 generation. (B) In the secondary screen, mosaic flies bearing large mutant clones in the wing were generated using hsFLP in conjunction with the Minute technique, and were screened for hh-like wing phenotypes. For details, see Results and Materials and Methods. The asterisk indicates a newly induced recessive mutation.

View larger version (95K): [in a new window]   Fig. 2. Eye phenotypes for mutations in the hh pathway. (A)Wild-type eye. (B-F) Eyes carrying mutant clones for smo3 (B), ttvl(2)00681 (C), hhSH2(D), dispSH23 (E) or cmnM82 (F). Loss-of-function mutations in the positive regulators of the hh pathway induce eye phenotypes similar to a hh mutation.

View larger version (77K): [in a new window]   Fig. 3. cmn affects Hh signaling. In this and the following figures, all the wing discs are oriented with anterior towards left and ventral up. (A) A wild-type wing and (B) a mosaic wing bearing large cmn mutant clones. The central structures of the wing including vein 3 and vein 4 are completely lost. Vein 2 and vein 5 are incomplete. The most anterior (anterior to vein 2) and most posterior (posterior to vein 5) structures are intact. (C,D) Third instar, wild-type (C) and cmn homozygous (D) wing discs were stained with anti-Dpp antibody. Dpp expression near the AP compartment boundary is diminished in the cmn mutant disc (indicated by the arrowheads). (E,F) Wild-type (E) and cmn homozygous (F) wing discs were stained with anti-Ptc antibody. Ptc upregulation is lost in the cmn mutant disc (arrowheads) (G,H). Wild-type (G) and cmn homozygous (H) wing discs were stained with anti-ß-gal antibody to visualize hh-lacZ expression. hh-lacZ expression appears normal in the cmn mutant disc.

View larger version (148K): [in a new window]   Fig. 4. cmn acts upstream of ptc. (A) Wild-type wing disc, (B) cmn homozygous wing disc and (C,D) cmn homozygous wing disc carrying a clone of ptc mutant cells, were stained to show dpp-lacZ reporter expression (red) and a marker gene (Myc) expression (green in D). ptc mutant cells are recognized by the lack of Myc expression. The cmn mutant disc exhibits diminished levels of dpp-lacZ expression (arrowhead in B). In contrast, cmn ptc double mutant cells situated in the anterior compartment express wild-type levels of dpp-lacZ (arrow in C and D) Arrowhead in C and D indicates dpp-lacZ expression at the AP boundary.

View larger version (143K): [in a new window]   Fig. 5. cmn is required in the posterior compartment. (A,A') A wing disc carrying a large clone of cmn mutant cells was stained to show the expression of ptc-lacZ (red) and a GFP marker gene (green). cmn mutant cells are identifiable by the lack of green staining. Anterior compartment cells near the AP compartment boundary express ptc-lacZ (arrowhead) at wild-type levels even though they are mutant for cmn. (B,B') A wing disc carrying a large clone of cmn mutant cells (marked by the lack of green staining) in the posterior compartment has lost ptc-lacZ expression in adjacent anterior compartment cells (arrowhead).

View larger version (117K): [in a new window]   Fig. 6. Hh distribution in wild-type and cmn mutant discs. (A,A',A'') A wild-type wing disc expressing UAS-Hh under the control of hh-Gal4 was stained to visualize Hh (red) and Ptc (green) protein distribution. Hh staining can be detected in anterior compartment cells near the AP compartment boundary. In these cells Hh colocalizes with Ptc in intracellular vesicles (arrows). (B,B',B'') A cmn homozygous mutant wing disc expressing UAS-Hh under the control of hh-Gal4 was stained to visualize Hh (red) and Ptc (green) distribution. Hh staining can only be detected in P-compartment cells, with little, if any, staining in A-compartment cells.

View larger version (165K): [in a new window]   Fig. 7. cmn mutant discs produce reduced levels of active Hh in the P-compartment. Late third instar wing discs were stained with anti-Col (green) or anti-Smo (red) antibody to monitor Hh signaling activity. (A-C). Wild-type wing discs activate col in A-compartment cells near the AP compartment boundary in the wing pouch region (A). disp mutant discs exhibit diminished levels of Col expression in a narrow stripe of A-compartment cells (arrowhead in B). Note that Col staining surrounding the wing pouch region is not controlled by Hh (indicated by asterisks in B). cmn mutant discs fail to express Col at detectable levels near the AP compartment boundary (C). (D-F) Expression of CiU in wild-type (D) or disp (E) discs activates Col expression at comparable levels in P-compartment cells, whereas expression of CiU in cmn mutant discs (F) induces ectopic Col expression in P-compartment cells at levels much lower than wild or disp mutant discs. (G-I). Wild-type (G) and disp (H) discs stabilized Smo at comparable levels in P-compartment cells whereas Smo was only slightly stabilized in P-compartment cells of cmn discs (I).

View larger version (124K): [in a new window]   Fig. 8. cmn is required for the activity of cholesterol-free Hh. Late third instar wing discs of the following genotypes were stained with anti-Ptc to monitor the up-regulation of ptc in response to Hh. (A) Wild type, (B) act>CD2>Gal4/UAS-HhN, (C) disp l(3)S037707, (D) act>CD2>Gal4/UAS-HhN; disp l(3)S037707, (E) cmnM82, (F) act>CD2>Gal4/UAS-HhN; cmnM82. Misexpressing HhN in both wild-type and disp mutant discs induced ectopic ptc upregulation (B,D). In contrast, misexpressing HhN in cmn mutant disc failed to upregulate ptc expression (F).

View larger version (44K): [in a new window]   Fig. 9. cmn mutations affect a putative membrane-bound acyltransferase. (A) Drosophila genomic sequence annotations around the cmnP630 P-element insertion site. Open boxes show nine annotated genes. Arrows indicate P-element insertion sites. (B) Schematic representation of the predicted protein encoded by CG11495, which is 500 aa. The 10 predicted transmembrane domains are underlined. Arrows mark the positions of the nonsense mutation we found in cmnM82 (aa 163), cmnMS6 (aa 163), cmnMS1 (aa 164), cmnM12 (aa 380) and cmnMS18 (aa 400). (C) The alignment of Cmn with several putative membrane-bound acyltransferases. Dm CMN, Drosophila cmn gene product; Sa DLTB, Staphylococcus DtlB; Hs ACAT, human cholesterol acyltransferase; Mm DGAT, mouse diacylglycerol O-acyltransferase; Dm PORC, Drosophila procupine gene product; Ce CAB16518, homolog of Cmn in the C. elegans genome. (D) Sequence alignment among Cmn and its human and mouse homologs. The underline indicates the region conserved among all members of the membrane-bound acyltransferase family.